Immunoassays have been used for many years in the detection of agents in samples. Such assays can be used to detect the presence of unwanted agents in a sample of fluid, for example the presence of bacteria in a bodily fluid or a pollutant in water. They can be used also to test for the presence of antibodies in body fluids, as an indication of the presence of an infectious agent, a cancer cell or an autoimmune disease.
Although many such assays represent extraordinary improvements over the prior methodology for determining the presence of an agent in a sample, the goal remains to develop immunoassays that are rapid and sensitive. It is a drawback of almost every immunoassay presently in use that the assay either is too slow (5-30 minutes) or, if faster, is not sufficiently sensitive (or both). Where the detection of disease is concerned, it is unacceptable to compromise on sensitivity, and accordingly those assays that are among the most sensitive tend to be cumbersome and slow.
Antibodies of the classes including IgA, IgG, or IgM specific for a particular antigen may be produced by animals at various disease stages. One or more classes prevail at different times. Ideally, the presence of all classes of antibodies specific for a particular antigen would be determined in a single assay. Such an assay would be highly sensitive because it would ensure detection of the presence of the prevalent class of antibody. However, heretofore known immunoassays do not achieve such sensitivity, due in large part to problems that result from unwanted specific binding and/or aggregation between species in test assay reagents, or unwanted binding between a test assay reagent and an analyte. To determine antibodies of a single class in a sample, assays have been developed that involve time-consuming analysis in which unwanted classes of antibodies are selectively removed from the sample solution prior to analysis of the antibody class of interest. Such a method is described in U.S. Pat. No. 4,829,012, issued May 9, 1989 to Cambiaso et al., in which the antibody class of interest is measured, after such selective removal, by inducing agglutination, and measuring the degree of agglutination. However, as a result of these problems, most assays measure the presence of only a single class of immunoglobulin, e.g. IgG.
One complication hindering development of an assay determinative of any or all of IgA, IgG, or IgM specific for a particular antigen in a single procedure is the fact that conventional proteins and antibodies that serve as binding partners for capturing antibodies of various classes also tend to bind to each other. Specifically, Protein A binds IgG, and this binding may be exploited in the determination of the presence of IgG. If the presence of IgA or IgM is to be determined, then anti-IgA or anti-IgM of the IgG class typically is utilized. The simultaneous determination of IgG using Protein A, and IgA or IgM using anti-IgA and/or anti-IgM of the IgG class according to heretofore known techniques, however, is complicated, as the assay reagents Protein A and anti-IgA or anti-IgM bind to each other. For that matter, the determination of any species in a sample using Protein A and a binding partner of the IgG class is complicated when performed according to known techniques.
Immunoassays also typically involve some kind of label for detecting the presence of the binding between an antibody and an analyte. Radioisotopes have commonly been used, but such use is discouraged for reasons including safety. Fluorophores, enzymes, chemiluminescent species, and the like also have been employed as labels in immunoassays. U.S. Pat. No. 4,373,932, issued Feb. 15, 1983 to Gribnau et al., describes reagents and test kits for the determination of immunochemically reactive components, in which one or more labeled species are attached to particles of an aqueous dispersion of a hydrophobic dye or pigment, or of polymer nuclei coated with such a dye or pigment. The use of such dyes offers many advantages, including safety and cost. The use of dyes in immunoassays, however, has not achieved widespread use perhaps because of problems with detection.